These data suggest an important role for JNK1 in innate immune responses

tro model of osteogenesis using adipose derived MSCs in monolayer and high-density cultures and present new evidence to show that resveratrol-activated Sirt-1 significantly favors osteogenic differentiation over adipogenic differentiation. The question of whether an interaction between resveratrol-activated Sirt-1 and Runx2 occurs 2 Resveratrol Promotes Osteogenesis of MSCs and whether this causes deacetylation of Runx2 during osteogenesis is an important focus of this study. Materials and Methods Antibodies Polyclonal anti-collagen type I antibody and alkaline phosphatase linked sheep anti-mouse and sheep anti-rabbit secondary antibodies for immunoblotting were purchased from Millipore. Polyclonal anti- Runx2 was purchased from Alpha Diagnostics Int. San Antonio, TX, USA. Monoclonal anti-b-actin and nicotinamide were purchased from SigmaAldrich. Polyclonal anti-Sirt-1 and antiNCoR were purchased from Abcam PLC. Polyclonal anti-PPAR-c antibodies were purchased from Acris Antibodies GmbH, Germany. Acetylated-lysine antibody was purchased from Cell Signaling Technology. Resveratrol with purity greater than 98% was purchased from Sigma-Aldrich. A 100-mM stock solution of 3 Resveratrol Promotes Osteogenesis of MSCs resveratrol was prepared in ethanol and further diluted in cell culture medium to prepare working concentrations. The maximum final content of ethanol in cultures was less than 0.1%. This concentration was also used as a control. Isolation and culture of 12829792 mesenchymal stem cells Mesenchymal stem cells were isolated from canine adipose tissue biopsies obtained during orthopedic surgeries, as previously described. Fully informed owner consent was obtained and the project was approved by the Ludwig-Maximilian University Ethical Review committee. Briefly, adipose tissue was cut into small pieces and digested with SB-366791 collagenase 0.2% in Ham’s-F12 in a water bath at 37uC for 2 hours. Digested adipose tissue was centrifuged at 1000 ” g/5 min and the pellet was resuspended in cell culture medium consisting of DMEM/Ham’s-F12 1:1, 10% FCS, 1% partricin solution, 1% penicillin/streptomycin solution, 75 mg/ml ascorbic acid, 1% essential amino acids and 1% Glutamine, all obtained from Seromed. The cells were seeded in a T75 cell culture flask and incubated at 37uC/5%CO2, 95% humidity. After four days, non-adherent cells were discarded by washing with Hank’s salt solution. The medium was changed three times per week. Adherent cells were split following formation of fibroblast-like cell colonies and upon reaching 6070% confluence, and were subcultured until the third or fourth passage was achieved. Pre-osteoblastic cell line culture. The mouse preosteoblastic cell line MC3T3-E1 was selected as an in vitro model of pre-osteoblastic cells, as previously described. The cells were cultured in alpha-MEM containing 10% FCS, 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were maintained in a humidified, 95% air/5% CO2 atmosphere at 37uC. All experiments were performed with third passage MC3T3-E1 cells. For induction of the osteoblast phenotype, cells were cultured in differentiation medium . Experimental design Osteogenic differentiation was performed in monolayer culture or in high-density mass culture. Mesenchymal stem cell cultures and pre-osteoblastic MC3T3-E1 cells were either left untreated, or incubated with one of the following treatments: 0.1, 1 and 10 mM resveratrol only; 1, 10 and 100 mM nicotinamide only; prestimulated with resveratrol